The invention relates generally to systems and methods for performing a reaction in a small volume without incurring an undesirable loss of the reaction volume due to evaporation, and more specifically to systems and methods for performing reactions involving polymers, particularly biopolymers, in a reaction volume of a few microliters or less in an unsealed environment.
Technological advances have allowed an examination of previously undiscernible phenomena. Such advances are particularly notable in the biological sciences, where the chemical and physical structures of many biopolymers have been described, and where such biopolymers, including DNA and proteins, routinely are synthesized and sequenced.
Although methods such as nucleic acid sequencing and synthesis have contributed to understanding the structure and function of biological molecules and their relationships to disease, the limitations of such methods are apparent. For example, it generally is agreed that knowledge of the entire sequence of the human genome would provide valuable insight into the prevention and treatment of disease. The human genome, however, contains over one billion nucleotides and a huge expenditure of labor and money would be required to sequence the entire human genome. Furthermore, using currently available methods, many years will be required to see the project to completion.
Similarly, it is a goal of most clinical researchers to develop rapid and simple tests for determining whether an individual has a disease or predisposition to a disease. In many cases, the signs and symptoms of many genetic diseases do not become apparent until an individual reaches a certain age or stage of development. Knowledge that an individual has a predisposition to a genetic disease can allow the clinician to take prophylactic measures to minimize or delay onset of the disease. Ideally, all individuals would be screened for potential genetically determined diseases, including screening a large number of genes in each individual. Unfortunately, such routine screening currently is not feasible because the assays are time consuming and the reagents for performing such assays are expensive and limited in availability.
In an effort to reduce the time and cost for analyzing biopolymers, including genes and proteins, processes are being developed to automate the analytic procedures. Automation provides a means for performing repetitive processes almost continually, except for periodic breaks for equipment maintenance, and allows researchers and technical staff to devote more time to other endeavors, including interpreting the results produced by the automated assays and trouble shooting problems that may arise. Automation of repetitive processes also provides the advantage that the likelihood of errors occurring, for example, due to fatigue or distraction is reduced and, therefore, more accurate results can be obtained.
The application of nanotechnology to the biological sciences promises to provide the next breakthroughs relating, for example, to the analysis, synthesis and utilization of biopolymers. Nanotechnology, which provides processes and apparatuses for performing procedures on a very small scale, has been developed by the semiconductor industry in order to produce smaller and smaller microchips, and to allow placement of a continually increasing number of instructions on a microchip.
Efforts are in progress to apply nanotechnology to chemical and biological procedures, thereby providing a means to perform assays in very small volumes, generally a few hundred nanoliters or less. Application of nanotechnology to biological assays can be particularly valuable because a critical limitation of many biological assays is the amount of biological material available for analysis. By performing such assays in nanoliter volumes or smaller, the effective concentration of a biopolymer in a biological reaction is increased, thereby providing the necessary kinetics for a biological reaction to proceed. In addition, the ability to perform biological assays in nanoliter volumes can provide a significant cost savings because much smaller amounts of reagents, which can be very expensive, can be utilized in the reactions.
The application of nanotechnology to biological assays has been hindered, in part, by the difficulty in manipulating and maintaining such small volumes. Many biological assays, for example, are performed in aqueous conditions, using water as a solvent, and at elevated temperatures, generally at least 37xc2x0 C., which is human body temperature. Water, like many liquid solvents, is susceptible to evaporation and, therefore, as the time or temperature of a reaction increases, the loss of water due to evaporation increases and the volume of the reaction decreases. As a result of evaporation, the effective concentration of reagents in the reaction increases, thereby changing the conditions of the reaction. Since most biological assays are quite sensitive to reaction conditions, loss of water or other solvent from a reaction can result in an assay that produces spurious results. Any loss of a solvent such as water is particularly deleterious when the reaction contains only a few hundred nanoliters or less of the liquid, since the reaction quickly can evaporate to dryness.
Various methods have been used to minimize the loss of solvent in a reaction due to evaporation in biochemical assays. For example, reaction mixtures can be drawn into glass capillary tubes, which then are sealed at both ends for the reaction. Small volume glass capillary tubes can be expensive, and the use of such tubes requires additional steps, including sealing and unsealing the tube, the latter which can produce glass shards.
In many cases, reaction mixtures are performed in a microcentrifuge tube or other open chamber, and evaporation is minimized by overlaying the reaction mixture with wax, mineral oil, or other nonvolatile compound during the reaction. Such a method, again, requires additional steps, including removing the sealing material following the reaction. In order to remove all or most of the sealing material, which can otherwise contaminate the sample and hinder further analysis, some loss of the sample being assayed inevitably occurs. Since most biological samples are limited to begin with, any loss of sample can preclude an interpretation of the results of the assay. In general, any additional manipulations of a sample will incur extra cost, either in terms of time or money, and loss or contamination of the sample.
More recently, biological reactions have been performed on microchips, which conveniently can be adapted to automated processes. Such microchips have been designed having a system including, for example, chambers, which hold the reactants, and channels, which connect the chambers and in which the reactants can be mixed and a reaction performed. Since the channels, in which the reaction occurs, provide a sealed or closed environment, there is little or no evaporative loss of the reaction volume. Thus far, however, the technology for preparing such a device allows for the placement of only one or few of such closed systems on a single microchip and, therefore, the number of reactions that can be performed at one time on a single chip is limited. Thus, a need exists for systems useful for performing reactions in a volume of a few microliters or less in an unsealed environment. Therefore it is an object here to provide systems and methods that satisfy this need and also provide additional advantages.

Systems are provided for performing a reaction in an unsealed environment. The systems are used for any desired reaction, including, but not limited to in situ biopolymer or polymer synthesis, such as nucleic acid and protein syntheses, protein and nucleic acid sequencing methods, such as oligonucleotide-based primer extension, nucleic acid amplification reactions, protein and nucleic acid protease- or nuclease-based degradations and others.
A system as disclosed herein is an open system for performing a reaction, such as a synthetic reaction or an assay, particularly in a submicroliter volume. The systems can include a support for performing the reaction; a nanoliter dispensing pipette for dispensing a submicroliter amount of a liquid to a target site on the support; a temperature controlling device for regulating the temperature of the surface of the support; and means for controlling the amount of liquid dispensed, where the amount of liquid dispensed corresponds to the amount of liquid evaporated from the support. A means for controlling the amount of liquid dispensed can include computer software that calculates the rate of evaporation and signals the dispensing pipette to deliver an amount of the liquid that corresponds to the amount lost due to evaporation. A means for controlling the amount of liquid dispensed also can be manual input, which can be performed by an individual.
A system as disclosed herein also can include a temperature measuring device for measuring the temperature of the surface of the support. The support can be any support having a surface, including, for example, a bead, pin, comb, wafer, well or microchip, and the support can be functionalized such that a substrate, for example, a biopolymer can be linked, either directly or indirectly via covalent or non-covalent interactions, to the support and immobilized.
An open system, as disclosed herein, also can include a solid support, which has a target site that can contain a volume of liquid, for example, a reaction mixture; a liquid dispensing system, which can dispense a liquid to the target site; a temperature controlling system, which can regulate the temperature of the solid support; and an interface, which can indicate an amount of liquid to be dispensed to the target site from the liquid dispensing system. An interface can include, for example, a computer using an appropriate algorithm. A computer can monitor the temperature of the solid support and, based on various parameters, including, for example, the chemical nature of the liquid, the surface area of the liquid exposed to the environment, and the time the liquid is maintained at a particular temperature, and can provide information as to the amount of liquid to be dispensed from the liquid dispensing system to the target site to maintain the liquid at a predetermined volume. Based on that information, the liquid dispensing system can be manipulated manually, to dispense the liquid to the target site, or can be controlled automatically, for example, by interfacing it with the computer. In a system as disclosed herein, the amount of liquid dispensed from a liquid dispensing system to a target site generally corresponds to an amount of liquid lost from the target site due to evaporation, although the amount added also can be an initial amount added to a target site or an amount added to modify the conditions of a reaction.
An open system, as disclosed herein, also can include a solid support having a target site; a liquid dispensing system, which can dispense a liquid to the target site; a temperature controlling system, which regulates the temperature of the solid support; and means for regulating an amount of liquid dispensed from the liquid dispensing system. In addition, an open system, as disclosed herein, can have a means for containing a reaction mixture; a means for dispensing a liquid; a means for controlling the temperature of the reaction volume containing means; and means for regulating an amount of liquid dispensed from the liquid dispensing means.
A means for regulating an amount of liquid dispensed can be a computer having an appropriate algorithm. Such a computer can interface with the solid support, thereby monitoring the temperature of the support, and can provide an indication of an amount of liquid to be dispensed to a target site to maintain a predetermined volume, for example, of a reaction volume. The computer can cause to be displayed the amount of liquid to be dispensed, such that an individual can manipulate the liquid dispensing system and dispense the liquid, or the computer can further interface with the liquid dispensing system, thereby causing the amount of liquid to be dispensed. In addition, charts can be developed that predict the amount and rate of evaporation of a particular solvent at a particular temperature and, based on such charts, an individual can manipulate the liquid dispensing system as necessary. Also, a decrease in the volume of a liquid due to evaporation can be identified directly by including the liquid in a circuit, wherein, when the liquid falls below a predetermined point, the circuit is broken, thereby indicating that a liquid should be dispensed to the target site until the circuit is reestablished.
Methods for maintaining a volume of a liquid in an unsealed environment also are provided. A method for performing a reaction in a predetermined submicroliter volume in the open can be performed by dispensing the predetermined submicroliter volume of liquid onto the surface of a support; optionally monitoring the temperature of the substrate; determining the amount or rate of evaporation of the liquid from the support; and dispensing a further amount of the liquid to the surface of the support, wherein the further amount dispensed corresponds to the amount lost from the support due to evaporation, thereby maintaining the reaction volume at a predetermined volume throughout the course of the reaction. Such a method also can be performed, for example, by determining the temperature of a solid support, which has a target site that can contain a volume of liquid; and, based on the temperature, dispensing at the target site an amount of liquid required to maintain a predetermined volume of the liquid. The amount of liquid to be dispensed can be determined using a computer algorithm, which, based on various parameters, including the temperature of the support, the chemical nature of the liquid, the surface area of the liquid exposed to the environment, and the volume to be maintained, can indicate the amount of liquid that evaporates from the site and, therefore, the amount of liquid to be dispensed to maintain a predetermined volume. The volume of a liquid on a target site also can be monitored, for example, by microscopic examination, using an appropriate optical system or a video imaging device, such that, as the volume of a liquid at a target site decreases due to evaporation, a corresponding amount of liquid can be dispensed to maintain the volume within acceptable parameters.
Methods for performing a reaction in an unsealed environment also are provided. Such a method can be performed, for example, by determining the temperature of a solid support, which has a target site containing a volume of the reaction mixture, or determining the rate or amount of evaporation of liquid from the reaction mixture; and dispensing into the reaction mixture an amount of liquid required to maintain the volume at a predetermined level. Such a method is particularly useful where the reaction mixture has a volume of a few microliters or less, particularly a volume of about 500 nanoliters or less. The disclosed methods also are useful for performing submicroliter reactions at temperatures where the vapor pressure of a liquid in the reaction mixture is undesirably high, for example, about 2.5 kiloPascals (kPa) or greater, particularly about 5 kPa or greater, or about 10 kPa or greater, such that evaporation of the liquid can substantially change the volume of the reaction mixture and adversely affect the reaction. As such, the disclosed methods are useful for performing various chemical, physical and biological reactions, for example, a polymerase chain reaction, or a nucleic acid or polypeptide synthesis or sequencing reaction or other reaction or assay performed on a solid support.